Detection of vitamins A and E by tandem mass spectrometry

ABSTRACT

Methods are described for measuring the amount of one or more of vitamin A, α-tocopherol, and the combination of β-tocopherol and γ-tocopherol in a sample. More specifically, mass spectrometric methods are described for detecting and quantifying one or more of vitamin A, α-tocopherol, and the combination of β-tocopherol and γ-tocopherol in a sample.

CROSS-REFERENCED TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e)to U.S. Provisional Application Ser. No. 61/383,280 filed Sep. 15, 2010,which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to the quantitative measurement of one or more ofvitamin A (retinol), α-tocopherol, and β-tocopherol and/or γ-tocopherol.In a particular aspect, the invention relates to methods forquantitative measurement of one or more of these vitamins by massspectrometry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

There are five major fat-soluble vitamins or vitamin related substancesin humans: vitamins A, K, D, and E, and carotene. Vitamin A (retinol) isthe immediate precursor of two important biologically activemetabolites: retinal (needed for scotopic and color vision), andretinoic acid (a hormone-like growth factor for epithelial and othercells). Thus retinol is associated with the function of vision,epithelial cell integrity, bone remodeling, and reproduction. Vitamin Eexists in eight different forms (isomers): alpha (α), beta (β), gamma(γ), and delta (δ)-tocopherol; and alpha (α), beta (β), gamma (γ), anddelta (δ) tocotrienol. Alpha-tocopherol is the most active form ofvitamin E in humans, and may be an important lipid-soluble antioxidantin the protection of cell membranes from oxidation from radicalsproduced in the oxidation of fats. Alpha-tocopherol is the most studiedof the vitamin E isomers, with all other forms much less studied.

Various methods have been reported in the art for measuring vitamin A,α-tocopherol, β-tocopherol and γ-tocopherol, either individually or invarious combinations. For reports of detection of vitamin A, see e.g.,Deuker, S., et al., Anal. Chem. 1994, 66:4177-85 (reporting quantitationof derivatized retinol by gas chromatography-mass spectrometry); Wang,Y., et al., J. Mass Spectrom. 2001, 36:882-88 (reporting quantitation ofvitamin A (retinol) in rat prostate with liquid chromatography-massspectrometry); Li H., et al., et al., J. Chromatog. B 2005, 816:49-56(reporting quantitation of all-trans-retinol in fish eggs by liquidchromatography-tandem mass spectrometry with positive electrosprayionization); Rühl, R., Rapid Commun. Mass Spectrom. 2006, 20:2497-2504(reporting quantitation of retinol in serum and cell extracts by liquidchromatography-diode-array detection atmospheric pressure chemicalionization tandem mass spectrometry). For reports of detection ofα-tocopherol and/or γ-tocopherol, see e.g., Walton, T., et al., Biomed.Environ Mass Spectrom. 1988, 16:289-98 (reporting fragmentation spectraof α and γ forms of tocopherol generated by tandem mass spectrometry ofpure samples); Lauridsen, C., et al., Anal. Biochem. 2001, 289:89-95(reporting quantitation of α-tocopherol in plasma by HPLC-tandem massspectrometry); Mottier, P., et al., Anal. Biochem. 2002, 301:128-135(reporting quantitation of α-tocopherol in plasma by GC-tandem massspectrometry and HPLC-tandem mass spectrometry); Hao, Z., et al., J.Chromatog. A 2005, 1094:83-90 (reporting quantitation of α-tocopherol inbotanical materials by liquid chromatography-tandem mass spectrometry);Stöggl, W., et al., J. Sep. Sci. 2005, 28:1712-18 (reporting reversephase liquid chromatographic separation of α-tocopherol and γ-tocopherolwith a C-30 chromatography column); and Nagy, K., et al., Anal. Chem.2007, 79:7087-96 (reporting quantitation α-tocopherol and γ-tocopherolin plasma with normal phase liquid chromatography-tandem massspectrometry). For reports of detection of two or more of vitamin A,α-tocopherol, and γ-tocopherol, see e.g., Khachik, F., et al., Anal.Chem. 1992, 64:2111-22 (reporting detection of vitamin A, α-tocopherol,and γ-tocopherol from extracts of human plasma with HPLC); Khachik, F.,et al., Anal. Chem. 1997, 69:1873-81 (reporting quantitation of vitaminA, α-tocopherol, and γ-tocopherol in serum and breast milk with highperformance liquid chromatography—photodiode array detection—massspectrometry); Heudi, O., et al., J. Chromatog. A 2004, 1022:115-23(reporting simultaneous quantitation of vitamin A and E (formunspecified) in infant formulae by liquid chromatography-massspectrometry); Andreoli, R., et al., Anal. Bioanal. Chem. 2004,378:987-94 (reporting simultaneous determination of vitamins A (retinol)and α-tocopherol in serum with liquid chromatography-tandem massspectrometry); Capote, F., et al., Rapid Commun. Mass Spectrom. 2007,21:1745-54 (reporting quatitation of liposoluble vitamins includingvitamin A (all-trans-retinol) and α-tocopherol in human serum withliquid chromatography-triple quadrupole tandem mass spectrometry); andKamao, M., et al., J. Chromatog. B 2004, 1022:115-23 (reportingquantitation of fat soluble vitamins (including vitamins A (retinol) andα-tocopherol) in breast milk by liquid chromatography-tandem massspectrometry).

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the presence oramount of one or more of vitamin A, α-tocopherol, and the combinedamount of β-tocopherol and γ-tocopherol in a sample by tandem massspectrometry. The methods include subjecting the sample to ionizationunder conditions suitable to produce one or more ions detectable by massspectrometry; determining the amount of said one or more ions by tandemmass spectrometry; and using the amount of the one or more ions todetermine the amount of one or more of vitamin A, α-tocopherol, and thecombined amount of β-tocopherol and γ-tocopherol in the sample.

In one aspect, the present invention provides methods for determiningthe amount of vitamin A in a sample by tandem mass spectrometry. Thesemethods include subjecting vitamin A from the sample to ionization underconditions suitable to produce one or more ions detectable by massspectrometry; determining the amount of one or more of the ions bytandem mass spectrometry; and using the determined amounts of the one ormore ions to determine the amount of vitamin A in the sample. In theseembodiments, tandem mass spectrometry includes fragmenting a vitamin Aprecursor ion with a mass to charge ratio of about 269.30±0.80 into oneor more fragment ions comprising a fragment ion with mass to chargeratio of about 105.00±0.80. In some embodiments, the ion masses for thevitamin A precursor and fragment ions are those indicated±0.50.

In some related embodiments, the sample is subjected to liquidchromatography (LC) (such as reverse phase LC) prior to ionization. Insome related embodiments, the LC is HPLC. In some related embodiments,the LC and ionization are conducted with on-line processing.

In some related embodiments, the methods are also used to simultaneouslydetermine the amount of one or more analytes in addition to vitamin A.In some related embodiments, these additional analytes include one orboth of α-tocopherol and combined β-tocopherol and γ-tocopherol. In someembodiments, the additional analytes include α-tocopherol and combinedβ-tocopherol and γ-tocopherol.

In some methods where α-tocopherol is determined by tandem massspectrometry, determining the amount of α-tocopherol includesfragmenting a α-tocopherol precursor ion with a mass to charge ratio ofabout 430.47±0.80 into one or more fragment ions comprising a fragmention with mass to charge ratio of about 165.03±0.80. In some embodiments,the ion masses for the α-tocopherol precursor and fragment ions arethose indicated±0.50.

In some embodiments where the combined amount of β-tocopherol andγ-tocopherol is determined by tandem mass spectrometry, determining thecombined amount of β-tocopherol and γ-tocopherol comprises fragmentingβ-tocopherol and/or γ-tocopherol precursor ions with a mass to chargeratio of about 416.35±0.80 into one or more fragment ions comprising afragment ion with mass to charge ratio of about 151.00±0.80. In someembodiments, the ion masses for the β-tocopherol and/or γ-tocopherolprecursor and fragment ions are those indicated±0.50.

In some embodiments, the sample comprises a biological sample; inrelated embodiments, the biological sample may be from a human patientsuspected of having a deficiency or an excess of vitamin A. In someembodiments the sample comprises serum, such as human serum or plasma,such as EDTA plasma.

In embodiments utilizing tandem mass spectrometry, tandem massspectrometry may be conducted by any method known in the art, includingfor example, multiple reaction monitoring, precursor ion scanning, orproduct ion scanning.

In embodiments which utilize two or more of an extraction column, ananalytical column, and an ionization source, two or more of thesecomponents may be connected in an on-line fashion to allow for automatedsample processing and analysis.

In certain preferred embodiments of the methods disclosed herein, massspectrometry is performed in positive ion mode. Alternatively, massspectrometry is performed in negative ion mode. Various ionizationsources, including for example atmospheric pressure chemical ionization(APCI) or electrospray ionization (ESI), may be used in embodiments ofthe present invention. In certain preferred embodiments, one or more ofvitamin A, α-tocopherol, and the combined β-tocopherol and γ-tocopherolare measured using APCI in positive ion mode.

In preferred embodiments, a separately detectable internal standard isprovided in the sample, the amount of which is also determined in thesample. In these embodiments, all or a portion of both the analyte(s) ofinterest and the internal standard present in the sample are ionized toproduce a plurality of ions detectable in a mass spectrometer, and oneor more ions produced from each are detected by mass spectrometry. Inthese embodiments, the presence or amount of ions generated from theanalyte(s) of interest may be related to the presence of amount ofanalyte of interest in the sample.

In other embodiments, the amount of one or more of vitamin A,α-tocopherol, and combined β-tocopherol and γ-tocopherol in a sample maybe determined by comparison to one or more external reference standards.Exemplary external reference standards include blank plasma or serumspiked with one or more of vitamin A, α-tocopherol, β-tocopherol, andγ-tocopherol or isotopically labeled variants thereof.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedprecursor or fragment ions by mass spectrometry. Relative reduction asthis term is used does not require that any substance, present with theanalyte of interest in the material to be purified, is entirely removedby purification.

As used herein, the term “sample” refers to any sample that may containan analyte of interest. As used herein, the term “body fluid” means anyfluid that can be isolated from the body of an individual. For example,“body fluid” may include blood, plasma, serum, bile, saliva, urine,tears, perspiration, and the like. In preferred embodiments, the samplecomprises a body fluid sample; preferably plasma or serum. In someembodiments, the body fluid is from a human patient, such as a humanpatient suspected of having a deficiency or an excess of one or more ofvitamin A, α-tocopherol, and β-tocopherol and/or γ-tocopherol.

As used herein, the term “simultaneous” as applied to simultaneouslyionizing and/or detecting the amount of two or more analytes from asample means ionizing two or more analytes and/or acquiring datareflective of the amount of the two or more analytes in the sample fromthe same sample injection. The data for each analyte may be acquiredsequentially or in parallel, depending on the instrumental techniquesemployed. For example, a single sample containing two analytes may beinjected into an on-line HPLC column, which may then elute each analyteone after the other, resulting in introduction of the analytes into amass spectrometer at two different times. Determining the amount of eachof these two analytes is simultaneous for the purposes herein, as bothanalytes result from the same sample injection into the HPLC.

As used herein, the term “solid phase extraction” or “SPE” refers to aprocess in which a chemical mixture is separated into components as aresult of the affinity of components dissolved or suspended in asolution (i.e., mobile phase) for a solid through or around which thesolution is passed (i.e., solid phase). In some instances, as the mobilephase passes through or around the solid phase, undesired components ofthe mobile phase may be retained by the solid phase resulting in apurification of the analyte in the mobile phase. In other instances, theanalyte may be retained by the solid phase, allowing undesiredcomponents of the mobile phase to pass through or around the solidphase. In these instances, a second mobile phase is then used to elutethe retained analyte off of the solid phase for further processing oranalysis. SPE, including TFLC, may operate via a unitary or mixed modemechanism. Mixed mode mechanisms utilize ion exchange and hydrophobicretention in the same column; for example, the solid phase of amixed-mode SPE column may exhibit strong anion exchange and hydrophobicretention; or may exhibit column exhibit strong cation exchange andhydrophobic retention.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC),normal phase liquid chromatography (NPLC), high performance liquidchromatography (HPLC), and turbulent flow liquid chromatography (TFLC)(sometimes known as high turbulence liquid chromatography (HTLC) or highthroughput liquid chromatography). As used herein, the term “reversephase liquid chromatography” refers to any liquid chromatographytechnique in which the mobile phase is polar and the stationary phase isnon-polar. As used herein, the term “normal phase liquid chromatography”refers to any liquid chromatography technique in which the mobile phaseis non-polar and the stationary phase is polar.

As used herein, the term “high performance liquid chromatography” or“HPLC” (sometimes known as “high pressure liquid chromatography”) refersto liquid chromatography in which the degree of separation is increasedby forcing the mobile phase under pressure through a stationary phase,typically a densely packed column.

As used herein, the term “turbulent flow liquid chromatography” or“TFLC” (sometimes known as high turbulence liquid chromatography or highthroughput liquid chromatography) refers to a form of chromatographythat utilizes turbulent flow of the material being assayed through thecolumn packing as the basis for performing the separation. TFLC has beenapplied in the preparation of samples containing two unnamed drugs priorto analysis by mass spectrometry. See, e.g., Zimmer et al., J ChromatogrA 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368,5,795,469, and 5,772,874, which further explain TFLC. Persons ofordinary skill in the art understand “turbulent flow”. When fluid flowsslowly and smoothly, the flow is called “laminar flow”. For example,fluid moving through an HPLC column at low flow rates is laminar. Inlaminar flow the motion of the particles of fluid is orderly withparticles moving generally in straight lines. At faster velocities, theinertia of the water overcomes fluid frictional forces and turbulentflow results. Fluid not in contact with the irregular boundary “outruns”that which is slowed by friction or deflected by an uneven surface. Whena fluid is flowing turbulently, it flows in eddies and whirls (orvortices), with more “drag” than when the flow is laminar. Manyreferences are available for assisting in determining when fluid flow islaminar or turbulent (e.g., Turbulent Flow Analysis: Measurement andPrediction, P. S. Bernard & J. M. Wallace, John Wiley & Sons, Inc.,(2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott,Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 40 μm, such as greater than about 50 μm. Asused in this context, the term “about” means±10%.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means±10%. In apreferred embodiment the analytical column contains particles of about 5μm in diameter.

As used herein, the terms “on-line” and “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged species; and (2) detecting themolecular weight of the charged species and calculating a mass-to-chargeratio. The compounds may be ionized and detected by any suitable means.A “mass spectrometer” generally includes an ionizer and an ion detector.In general, one or more molecules of interest are ionized, and the ionsare subsequently introduced into a mass spectrometric instrument where,due to a combination of magnetic and electric fields, the ions follow apath in space that is dependent upon mass (“m”) and charge (“z”). See,e.g., U.S. Pat. No. 6,204,500, entitled “Mass Spectrometry FromSurfaces;” U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus forTandem Mass Spectrometry;” U.S. Pat. No. 6,268,144, entitled “DNADiagnostics Based On Mass Spectrometry;” U.S. Pat. No. 6,124,137,entitled “Surface-Enhanced Photolabile Attachment And Release ForDesorption And Detection Of Analytes;” Wright et al., Prostate Cancerand Prostatic Diseases 1999, 2: 264-76; and Merchant and Weinberger,Electrophoresis 2000, 21: 1164-67.

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber. As the droplets get smaller the electrical surface chargedensity increases until such time that the natural repulsion betweenlike charges causes ions as well as neutral molecules to be released.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for theionization of molecule M is photon absorption and electron ejection toform the molecular ion M+. Because the photon energy typically is justabove the ionization potential, the molecular ion is less susceptible todissociation. In many cases it may be possible to analyze sampleswithout the need for chromatography, thus saving significant time andexpense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase. Laser desorption thermal desorption is a technique wherein asample containing the analyte is thermally desorbed into the gas phaseby a laser pulse. The laser hits the back of a specially made 96-wellplate with a metal base. The laser pulse heats the base and the heatcauses the sample to transfer into the gas phase. The gas phase sampleis then drawn into the mass spectrometer.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, the term “lower limit of quantification”, “lower limitof quantitation” or “LLOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a relative standarddeviation (RSD %) of less than 20% and an accuracy of 85% to 115%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as three times the RSD ofthe mean at the zero concentration.

As used herein, an “amount” of an analyte in a body fluid sample refersgenerally to an absolute value reflecting the mass of the analytedetectable in volume of sample. However, an amount also contemplates arelative amount in comparison to another analyte amount. For example, anamount of an analyte in a sample can be an amount which is greater thana control or normal level of the analyte normally present in the sample.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to ±0.80 atomic mass unit, such as ±0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B show exemplary precursor and fragmentation MS/MS spectra,respectively, for vitamin A. Details are discussed in Example 3.

FIGS. 2A and B show exemplary precursor and fragmentation MS/MS spectra,respectively, for α-tocopherol. Details are discussed in Example 3.

FIGS. 3A and B show exemplary precursor and fragmentation MS/MS spectra,respectively, for β-tocopherol and/or γ-tocopherol. Details arediscussed in Example 3.

FIGS. 4A and B show exemplary precursor and fragmentation MS/MS spectra,respectively, for d₉-α-tocopherol. Details are discussed in Example 3.

FIG. 5 shows a plot of the results from the lower linearity ofquantitation studies for vitamin A. The data plotted with squaresrepresent the accuracy (%); data plotted with triangles representrelative standard deviation (RSD %); and data plotted with diamondsrepresent concentration. Details are described in Example 4.

FIG. 6 shows a plot of the results from the lower linearity ofquantitation studies for α-tocopherol. The data plotted with squaresrepresent the accuracy (%); data plotted with triangles representrelative standard deviation (RSD %); and data plotted with diamondsrepresent concentration. Details are described in Example 4.

FIG. 7 shows a plot of the results from the lower linearity ofquantitation studies for γ-tocopherol. The data plotted with squaresrepresent the accuracy (%); data plotted with triangles representrelative standard deviation (RSD %); and data plotted with diamondsrepresent concentration. Details are described in Example 4.

FIG. 8 shows a plot of the linearity of quantitation of vitamin A.Details are described in Example 5.

FIG. 9 shows a plot of the linearity of quantitation of α-tocopherol.Details are described in Example 5.

FIG. 10 shows a plot of the linearity of quantitation of β-tocopheroland/or γ-tocopherol. Details are described in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for measuring the amount of one or more ofvitamins A (retinol), α-tocopherol, and β-tocopherol and/or γ-tocopherolin a sample. More specifically, mass spectrometric methods are describedfor detecting and quantifying vitamin A, α-tocopherol, and β-tocopheroland/or γ-tocopherol in a sample. The methods may utilize liquidchromatography to perform a purification of selected analytes, combinedwith methods of mass spectrometry (MS), thereby providing ahigh-throughput assay system for detecting and quantifying the amount ofone or more of vitamin A (retinol), α-tocopherol, and β-tocopherol andγ-tocopherol in a sample. The preferred embodiments are particularlywell suited for application in large clinical laboratories for automatedvitamin A (retinol), α-tocopherol, and β-tocopherol and γ-tocopherolquantification assay.

β-tocopherol and γ-tocopherol are positional isomers, each with a molarmass of about 416.88 a.m.u. In certain embodiments, ionization ofβ-tocopherol and γ-tocopherol under similar conditions will generateβ-tocopherol and γ-tocopherol precursor ions with similar m/z. Further,tandem mass spectrometric methods which fragment similar β-tocopheroland γ-tocopherol precursor ions may result in generation of similarβ-tocopherol and γ-tocopherol fragment ions. Thus in some embodiments,precursor and fragment ions from β-tocopherol may not be distinguishedfrom precursor and fragment ions from γ-tocopherol. Thus, in someembodiments, the amount of particular precursor and fragment ions maycorrelate to the combined amount of β-tocopherol and/or γ-tocopherol ina sample.

Similarly, certain purification methods used in some of the methodsdescribed herein are unable to separate β-tocopherol and γ-tocopherol.For example, β-tocopherol and γ-tocopherol in a sample will co-elutewhen the sample is purified by certain liquid chromatography procedures.Thus, in some methods, β-tocopherol and γ-tocopherol will be introducedinto the mass spectrometer at the same time if both β-tocopherol andγ-tocopherol are present in the sample.

Suitable test samples for use in methods of the present inventioninclude any test sample that may contain the analyte of interest. Insome preferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Preferred samples comprise bodilyfluids such as blood, plasma, serum, saliva, cerebrospinal fluid, ortissue samples; preferably plasma and serum. Such samples may beobtained, for example, from a patient; that is, a living person, male orfemale, presenting oneself in a clinical setting for diagnosis,prognosis, or treatment of a disease or condition. In embodiments wherethe sample comprises a biological sample, the methods may be used todetermine the amount of one or more of vitamin A, α-tocopherol, andβ-tocopherol and γ-tocopherol in the sample when the sample was obtainedfrom the biological source (i.e., the amount of endogenous vitamin A,α-tocopherol, and β-tocopherol and γ-tocopherol in the sample).

The present invention also contemplates kits for a vitamin A,α-tocopherol, and β-tocopherol and γ-tocopherol quantitation assay. Akit for a vitamin A, α-tocopherol, and β-tocopherol and γ-tocopherolquantitation assay may include a kit comprising the compositionsprovided herein. For example, a kit may include packaging material andmeasured amounts of an isotopically labeled internal standard, inamounts sufficient for at least one assay. Typically, the kits will alsoinclude instructions recorded in a tangible form (e.g., contained onpaper or an electronic medium) for using the packaged reagents for usein a vitamin A, α-tocopherol, and a combination of β-tocopherol andγ-tocopherol quantitation assay.

Calibration and QC pools for use in embodiments of the present inventionare preferably prepared using a matrix similar to the intended samplematrix, provided that the analyte(s) of interest (i.e., vitamin A,α-tocopherol, β-tocopherol, and/or γ-tocopherol) are essentially absent.

Sample Preparation for Mass Spectrometric Analysis

In preparation for mass spectrometric analysis, one or more of vitaminA, α-tocopherol, β-tocopherol, and γ-tocopherol may be enriched relativeto one or more other components in the sample (e.g. protein) by variousmethods known in the art, including for example, liquid chromatography,filtration, centrifugation, thin layer chromatography (TLC),electrophoresis including capillary electrophoresis, affinityseparations including immunoaffinity separations, extraction methodsincluding ethyl acetate, methanol, or ethanol extraction, and the use ofchaotropic agents or any combination of the above or the like.

Specifically, test samples (and in particular, biological samples suchas samples comprising serum) may be subjected to liquid-liquidextraction as an initial step. Internal standard is typically added tothe test samples prior to liquid-liquid extraction. In certainembodiments, the test samples are subjected to liquid-liquid extractionby mixing with equal amounts of absolute ethanol and hexanes. Therelative volumes of the solvents to the volume of a test sample iseasily determined by one in the art and may be about 300 microliters oftest sample to about 1 ml of ethanol and 1 ml of hexanes.

Protein precipitation is another method of preparing a test sample,especially a biological test sample, such as serum. Protein purificationmethods are well known in the art, for example, Polson et al., Journalof Chromatography B 2003, 785:263-275, describes protein precipitationtechniques suitable for use in methods of the present invention. Proteinprecipitation may be used to remove most of the protein from the sampleleaving one or more of vitamin A (retinol), α-tocopherol, β-tocopherol,and γ-tocopherol in the supernatant. The samples may be centrifuged toseparate the liquid supernatant from the precipitated proteins;alternatively the samples may be filtered to remove precipitatedproteins. The resultant supernatant or filtrate may then be applieddirectly to mass spectrometry analysis; or alternatively to liquidchromatography and subsequent mass spectrometry analysis. In certainembodiments, the use of protein precipitation such as for example,formic acid protein precipitation, may obviate the need for TFLC orother on-line extraction prior to mass spectrometry or HPLC and massspectrometry.

Another method of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Certain methods of liquidchromatography, including HPLC, rely on relatively slow, laminar flowtechnology. Traditional HPLC analysis relies on column packing in whichlaminar flow of the sample through the column is the basis forseparation of the analyte of interest from the sample. The skilledartisan will understand that separation in such columns is a partitionprocess and may select LC, including HPLC, instruments and columns thatare suitable for use with vitamin A (retinol), α-tocopherol,β-tocopherol, and γ-tocopherol. The chromatographic column typicallyincludes a medium (i.e., a packing material) to facilitate separation ofchemical moieties (i.e., fractionation). The medium may include minuteparticles. The particles typically include a bonded surface thatinteracts with the various chemical moieties to facilitate separation ofthe chemical moieties. One suitable bonded surface is a hydrophobicbonded surface such as an alkyl bonded or a cyano bonded surface. Alkylbonded surfaces may include C-4, C-8, C-12, or C-18 bonded alkyl groups.In preferred embodiments, the column is a reversed phase C-12 column.The chromatographic column includes an inlet port for receiving a sampleand an outlet port for discharging an effluent that includes thefractionated sample. The sample may be supplied to the inlet portdirectly, or from a SPE column, such as an on-line extraction column ora TFLC column.

In one embodiment, the sample may be applied to the LC column at theinlet port, eluted with a solvent or solvent mixture, and discharged atthe outlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytypic (i.e.mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, HPLC is conducted with a reversed phaseanalytical column chromatographic system. In certain preferredembodiments, a reversed phase C-12 analytical column (e.g., a SynergiMax-RP C-12 analytical column from Phenomenex Inc. (4 μm particle size,70×3.0 mm), or equivalent) is used. In certain preferred embodiments,HPLC is performed with an isocratic flow comprising about 50% HPLC Grademethanol, about 30% acetonitrile, and about 20% dichloromethane.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, TFLC may be used for purification of vitamin A(retinol), α-tocopherol, and γ-tocopherol prior to mass spectrometry. Insuch embodiments, samples may be extracted using a TFLC column whichcaptures the analyte. The analyte is then eluted and transferred on-lineto an analytical HPLC column. For example, sample extraction may beaccomplished with a TFLC extraction cartridge may be accomplished with alarge particle size (50 μm) packed column. Sample eluted off of thiscolumn is then transferred on-line to an HPLC analytical column forfurther purification prior to mass spectrometry. Because the stepsinvolved in these chromatography procedures may be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized. This feature may result insavings of time and costs, and eliminate the opportunity for operatorerror.

Detection and Quantitation by Mass Spectrometry

In various embodiments, vitamin A, α-tocopherol, and γ-tocopherol may beionized by any method known to the skilled artisan. Mass spectrometry isperformed using a mass spectrometer, which includes an ion source forionizing the fractionated sample and creating charged molecules forfurther analysis. For example ionization of the sample may be performedby electron ionization, chemical ionization, electrospray ionization(ESI), photon ionization, atmospheric pressure chemical ionization(APCI), photoionization, atmospheric pressure photoionization (APPI),Laser diode thermal desorption (LDTD), fast atom bombardment (FAB),liquid secondary ionization (LSI), matrix assisted laser desorptionionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, surface enhanced laser desorptionionization (SELDI), and particle beam ionization. The skilled artisanwill understand that the choice of ionization method may be determinedbased on the analyte to be measured, type of sample, the type ofdetector, the choice of positive versus negative mode, etc.

Vitamin A, α-tocopherol, β-tocopherol and γ-tocopherol may be ionized inpositive or negative mode. In preferred embodiments, vitamin A,α-tocopherol, β-tocopherol and γ-tocopherol are ionized by APCI inpositive ion mode.

In mass spectrometry techniques generally, after the sample has beenionized, the positively or negatively charged ions thereby created maybe analyzed to determine a mass-to-charge ratio. Suitable analyzers fordetermining mass-to-charge ratios include quadrupole analyzers, iontraps analyzers, and time-of-flight analyzers. Exemplary ion trapmethods are described in Bartolucci, et al., Rapid Commun. MassSpectrom. 2000, 14:967-73.

The ions may be detected using several detection modes. For example,selected ions may be detected, i.e. using a selective ion monitoringmode (SIM), or alternatively, mass transitions resulting from collisioninduced dissociation or neutral loss may be monitored, e.g., multiplereaction monitoring (MRM) or selected reaction monitoring (SRM).Preferably, the mass-to-charge ratio is determined using a quadrupoleanalyzer. For example, in a “quadrupole” or “quadrupole ion trap”instrument, ions in an oscillating radio frequency field experience aforce proportional to the DC potential applied between electrodes, theamplitude of the RF signal, and the mass/charge ratio. The voltage andamplitude may be selected so that only ions having a particularmass/charge ratio travel the length of the quadrupole, while all otherions are deflected. Thus, quadrupole instruments may act as both a “massfilter” and as a “mass detector” for the ions injected into theinstrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

Alternate modes of operating a tandem mass spectrometric instrumentinclude product ion scanning and precursor ion scanning For adescription of these modes of operation, see, e.g., E. Michael Thurman,et al., Chromatographic-Mass Spectrometric Food Analysis for TraceDetermination of Pesticide Residues, Chapter 8 (Amadeo R.Fernandez-Alba, ed., Elsevier 2005) (387).

The results of an analyte assay may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, external standards may be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion may be converted into an absolute amount of the originalmolecule. In certain embodiments, an internal standard is used togenerate a standard curve for calculating the quantity of one or more ofvitamin A (retinol), α-tocopherol, and the combination of β-tocopheroland γ-tocopherol. Methods of generating and using such standard curvesare well known in the art and one of ordinary skill is capable ofselecting an appropriate internal standard. For example, in preferredembodiments one or more isotopically labeled fat soluble vitamin may beused as internal standards. For example, d₉-α-tocopherol or d₅-vitamin Amay be used as internal standards in the instant methods. Numerous othermethods for relating the amount of an ion to the amount of the originalmolecule will be well known to those of ordinary skill in the art.

As used herein, an “isotopic label” produces a mass shift in the labeledmolecule relative to the unlabeled molecule when analyzed by massspectrometric techniques. Examples of suitable labels include deuterium(²H)_(,) ¹³C, and ¹⁵N. One or more isotopic labels can be incorporatedat one or more positions in the molecule and one or more kinds ofisotopic labels can be used on the same isotopically labeled molecule.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activated dissociation (CAD) isoften used to generate fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In particularly preferred embodiments, one or more of vitamin A,α-tocopherol, and γ-tocopherol in a sample are detected and/orquantified using MS/MS as follows. Samples are preferably subjected toliquid-liquid extraction, the supernatant dried and reconstituted, thensubjected to liquid chromatography, preferably HPLC; the flow of liquidsolvent from a chromatographic column enters the heated nebulizerinterface of an MS/MS analyzer; and the solvent/analyte mixture issprayed at a high flow rate (such as between about 0.4 and 1 ml/min) toform an aerosol cloud, which is subjected to a corona discharge. Duringthese processes, the analyte or analytes (i.e., one or more of vitaminA, α-tocopherol, β-tocopherol and γ-tocopherol) are ionized. The ions,e.g. precursor ions, pass through the orifice of the instrument andenter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are massfilters, allowing selection of ions (i.e., selection of “precursor” and“fragment” ions in Q1 and Q3, respectively) based on their mass tocharge ratio (m/z). Quadrupole 2 (Q2) is the collision cell, where ionsare fragmented. The first quadrupole of the mass spectrometer (Q1)selects for ions with the mass to charge ratios of precursor ions fromthe analyte or analytes (i.e., one or more of vitamin A, α-tocopherol,and γ-tocopherol). Precursor ions with the correct mass/charge ratiosare allowed to pass into the collision chamber (Q2), while unwanted ionswith any other mass/charge ratio collide with the sides of thequadrupole and are eliminated. Precursor ions entering Q2 collide withneutral argon gas molecules and fragment. The fragment ions generatedare passed into quadrupole 3 (Q3), where the fragment ions of theanalyte or analytes (i.e., one or more of vitamin A, α-tocopherol,β-tocopherol and γ-tocopherol) are selected while other ions areeliminated.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably positive ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of each analyte to bequantitated that may be used for selection in quadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC-MS methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, may be measured andcorrelated to the amount of the analyte of interest. In certainembodiments, the area under the curves, or amplitude of the peaks, forfragment ion(s) and/or precursor ions are measured to determine theamount of one or more of vitamin A, α-tocopherol, and combination ofβ-tocopherol and γ-tocopherol. As described above, the relativeabundance of a given ion may be converted into an absolute amount of theoriginal analyte using calibration standard curves based on peaks of oneor more ions of an internal molecular standard.

The lower limits of quantitation (LLOQ) is the point where measurementsbecome quantitatively meaningful. The analyte response at this LLOQ isidentifiable, discrete and reproducible with an imprecision (i.e.,relative standard deviation, or RSD) of equal to or less than 20% and anaccuracy of 85% to 115%. Methods of the present invention are capable ofachieving (LLOQ) for vitamin A of between about 10 mg/dL and 1.48 mg/dL,such as between about 5 mg/dL and 1.48 mg/dL, such as about 1.48 mg/dL;for α-tocopherol of between about 1.00 mg/L and 0.19 mg/L, such asbetween 0.50 mg/L and 0.19 mg/L, such as about 0.19 mg/L; and forcombined β-tocopherol and γ-tocopherol of between about 1.00 mg/L and0.12 mg/L, such as about 0.50 mg/L and 0.12 mg/L, such as about 0.12mg/L.

The limit of detection (LOD) is the point at which a value is beyond theuncertainty associated with its measurement and is defined as threestandard deviations from lowest measurable concentration. Methods of thepresent invention are capable of achieving a limit of detection (LOD)for vitamin A of between about 2.00 mcg/dL and 0.38 mcg/dL, such asbetween about 1.00 mcg/dL and 0.38 mcg/dL, such as about 0.38 mcg/dL;for α-tocopherol of between about 0.50 mg/L and 0.03 mg/L, such asbetween 0.25 mg/L and 0.03 mg/L, such as about 0.03 mg/L; and forcombined β-tocopherol and γ-tocopherol of between about 0.50 mg/L and0.03 mg/L, such as about 0.25 mg/L and 0.03 mg/L, such as about 0.03mg/L.

The following Examples serve to illustrate the invention. These Examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1: Sample Preparation

Standard stock solutions containing various amounts of vitamin A, stocksolutions containing various amounts of α-tocopherol, and stocksolutions containing various amounts of γ-tocopherol were prepared byspiking vitamin A, α-tocopherol, and γ-tocopherol in absolute ethanol.Internal standard solutions were prepared as above, but with d₅-retinol(i.e., d₅-vitamin A) and d₉-α-tocopherol at concentrations of about 2.5mcg/mL and 5.0 mcg/mL, respectively. Portions of the standard stocksolutions were spiked in analyte-stripped, defibrinated and delipidizedserum from Biocell (“Biocell serum”) for use as quality control samples(details discussed below in Example 4).

Chilled serum samples were prepared for analysis by warming to roomtemperature and vortexing for about 5-10 seconds. All stock standardsand controls were vortexed at the same time. After vortexing, stockstandards, controls, and serum samples were inspected for homogeneity.The remaining sample preparation steps were conducted with the mainlight in the room turned off.

Internal standard working solution was then added to all samples, alongwith about 1.0 ml of absolute ethanol and 1.0 ml of hexanes. The sampleswere then mixed for about 60-80 seconds, and centrifuged at 3000-3100RPM (at about 4° C.) for between 5 and 8 minutes.

The resulting supernatant hexane layers were collected from each sampleand transferred to a new glass tube, and evaporated to dryness underflowing nitrogen. The temperature during evaporation was about 20° C.and the evaporation was conducted under darkness. Once the solvent hasbeen evaporated, about 1.0 mL of mobile phase (about 50% HPLC Grademethanol, about 30% acetonitrile, and about 20% dichloromethane) wasadded to each sample. The resulting mixtures were then vortexed forabout 20-25 seconds and transferred to 96-well plates for LC-MS/MSanalysis.

Example 2: Extraction of Vitamin A, α-Tocopherol, and γ-Tocopherol fromSamples with Liquid Chromatography

Injection of about 20 μL of sample was performed with a CohesiveTechnologies Aria TLX-4 system using Aria OS V 1.5.1 or newer software.

The 20 μL sample injections were introduced into a Phenomenex SynergiMax-RP 4 μm (75×3.0 mm) analytical column. Alternatively, some sampleswere introduced into an Waters Atlantis T3 3 μm (50×2.1 mm) analyticalcolumn, used as a backup column. An isocratic HPLC mobile phase of about50% HPLC Grade methanol, about 30% acetonitrile, and about 20%dichloromethane was applied to the analytical column, to separate theanalytes from other species contained in the sample.

The separated analytes were then subjected to MS/MS for quantitation ofvitamin A, α-tocopherol, and γ-tocopherol from each sample injection.

Example 3: Detection of Vitamin A, α-Tocopherol, and γ-Tocopherol byTandem MS

MS/MS was performed using a Finnigan TSQ Quantum Ultra MS/MS system(Thermo Electron Corporation). The following software programs, all fromThermo Electron, were used in the Examples described herein: TSQ UltraQuantum V 1.4.1 or newer, Xcalibur V 2.0 or newer, and LCQuan V 2.5 ornewer. Liquid solvent/analyte exiting the analytical column flowed tothe APCI interface of the MS/MS analyzer and ionized.

Ions passed to the first quadrupole (Q1), which selected precursor ionswith a m/z of about 269.30±0.50 for vitamin A, a m/z of about274.20±0.50 for d₅-retinol, a m/z of about 430.47±0.50 for α-tocopherol,and a m/z of about 416.35±0.50 for γ-tocopherol, and a m/z of about439.50±0.50 for d₉-α-tocopherol. Ions entering quadrupole 2 (Q2)collided with argon gas (at a collision cell energy of between about 34and 47 V) to generate ion fragments, which were passed to quadrupole 3(Q3) for further selection. Mass transitions used for detection andquantitation during validation on positive polarity are listed inTable 1. The quantitation of all three analytes was accomplishedsimultaneously for each sample injection.

TABLE 1 Mass Transitions Monitored for Vitamin A, α-Tocopherol, andγ-Tocopherol, and Internal Standards (Positive Polarity) AnalytePrecursor Ion (m/z) Product Ions (m/z) Vitamin A 269.30 ± 0.50 105.00 ±0.50 d₅-Retinol 274.20 ± 0.50  93.10 ± 0.50 α-Tocopherol 430.47 ± 0.50165.03 ± 0.50 γ-Tocopherol 416.35 ± 0.50 151.00 ± 0.50 d₉-α-Tocopherol439.50 ± 0.50 174.10 ± 0.50

Several other potential precursor ions and product ions (for the abovedescribed precursor ions) were observed and could be used to supplementor replace any of the above indicated ions. Supplemental or alternateions can be seen for Vitamin A, α-Tocopherol, and γ-Tocopherol in theexemplary spectra shown in FIGS. 1A-B, 2A-B, and 3A-B, respectively.

Example 4: Analytical Sensitivity: Lower Limit of Quantitation (LLOQ)and Limit of Detection (LOD) for Vitamin A, α-Tocopherol, andγ-Tocopherol

The quantitation of vitamin A, α-tocopherol, and γ-tocopherol viamonitoring the indicated transitions with a triple quadrupole tandemmass spectrometer was conducted on spiked Biocell serum samples andpatient serum samples.

The LLOQ is the point where measurements become quantitativelymeaningful. The analyte response at this LLOQ is identifiable, discreteand reproducible with an imprecision (i.e., relative standard deviation,or RSD) of equal to or less than 20% and an accuracy of 85% to 115%. TheLLOQ was determined by assaying six levels of vitamin A, α-tocopherol,and γ-tocopherol (10 replicates for five days at each level), thendetermining the reproducibility.

Analysis of data for vitamin A (shown in Table 2) shows that serumspecimens in a concentration range of about 0.56 mcg/mL to about 11.8mcg/mL yield relative standard deviations of 10.9% to 42.2% andaccuracies of about 93.0% to about 158.6%. Acceptable reproducibilityconcentrations (RSD≤20%) are observed at 1.48 mg/dL and higher. The RSDand accuracy at low concentration levels is shown in FIG. 6.

TABLE 2 LLOQ Data for Spiked Vitamin A Serum Samples Vitamin A Standard1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Actual (mcg/dL)0.56 0.74 1.48 2.95 5.90 11.80 Replicate 1 0.56 0.70 1.56 3.84 6.4511.39 2 1.20 0.45 1.34 3.51 6.62 11.47 3 1.00 0.59 1.49 3.43 5.37 13.044 0.82 0.61 1.52 3.35 6.81 13.13 5 1.16 0.59 1.74 3.47 6.29 9.60 6 0.350.98 1.34 3.27 6.93 11.85 7 1.28 0.83 1.34 3.01 5.11 10.07 8 0.76 0.581.38 2.99 5.98 11.04 9 1.06 0.79 1.41 3.63 5.80 12.43 10  0.64 0.91 1.482.78 5.28 12.73 Replicate 1 0.59 0.67 1.20 3.52 5.18 10.21 2 0.42 0.711.33 2.74 4.83 10.50 3 0.61 0.84 1.60 2.74 7.40 11.03 4 1.21 0.42 1.132.61 5.05 10.39 5 0.56 0.80 1.32 2.50 7.13 9.49 6 0.78 0.34 1.21 2.606.64 9.29 7 0.56 0.64 1.28 3.06 5.94 11.15 8 1.45 0.85 1.17 2.57 6.209.89 9 0.99 0.51 1.01 2.66 5.10 11.72 10  1.01 0.82 1.45 2.98 5.01 10.77Replicate 1 1.48 0.94 1.55 2.73 4.65 12.18 2 1.18 0.99 1.13 2.40 4.7611.54 3 0.60 1.10 1.49 3.27 4.58 12.37 4 0.93 0.83 1.34 3.11 3.98 12.855 0.71 0.83 1.47 2.36 3.85 12.54 6 1.29 0.85 1.78 2.31 4.81 12.33 7 1.090.72 1.54 2.62 4.32 12.19 8 0.63 0.78 1.59 2.80 4.44 9.28 9 0.78 0.821.98 2.40 4.56 13.74 10  1.77 0.65 1.21 2.43 5.64 9.32 Replicate 1 1.020.52 1.22 2.62 7.15 12.80 2 0.54 0.03 1.20 3.81 6.88 12.95 3 1.20 0.251.48 3.10 7.13 9.85 4 1.02 0.20 1.39 2.61 7.47 10.67 5 0.99 0.27 1.413.18 6.18 10.53 6 0.66 0.05 1.25 2.63 7.58 11.36 7 1.00 0.09 1.38 2.977.14 12.59 8 1.01 0.18 1.40 3.01 5.27 11.36 9 0.90 0.58 1.76 3.57 5.5411.10 10  0.84 0.34 1.06 3.18 5.73 9.84 Replicate 1 1.21 0.90 1.46 2.775.38 11.93 2 0.47 1.08 1.46 2.51 5.46 11.10 3 0.20 0.79 1.48 2.68 5.2511.10 4 1.08 0.95 1.60 2.46 5.81 9.68 5 1.53 1.03 1.72 2.42 5.37 9.68 60.55 1.09 1.57 2.61 5.24 9.71 7 0.81 0.84 1.46 2.68 5.55 9.85 8 0.670.87 1.73 3.26 5.55 10.33 9 0.62 1.14 1.64 3.24 5.39 10.02 10  0.63 1.081.76 3.08 5.67 11.38 Average 0.89 0.69 1.44 2.92 5.71 11.15 SD 0.33 0.290.20 0.41 0.95 1.22 RSD (%) 37.3 42.4 14.3 14.0 16.7 10.9 Accuracy (%)158.6 93.0 97.0 99.0 96.8 94.5

Analysis of data for α-tocopherol (shown in Table 3) shows that serumspecimens in a concentration range of about 0.19 mg/L to about 4.00 mg/Lyield relative standard deviations of 6.3% to 13.4% and accuracies ofabout 92.6% to about 104.0%. Acceptable reproducibility concentrations(RSD≤20%) are observed at 0.19 mg/L and higher. The RSD and accuracy atlow concentration levels is shown in FIG. 7.

TABLE 3 LLOQ Data for Spiked α-Tocopherol Serum Samples α-TocopherolStandard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Actual(mg/L) 0.19 0.25 0.50 1.00 2.00 4.00 Replicate 1 0.18 0.28 0.48 1.052.15 4.32 2 0.19 0.26 0.52 1.10 2.22 4.36 3 0.19 0.24 0.48 1.10 2.184.57 4 0.19 0.26 0.52 1.18 2.26 4.51 5 0.20 0.27 0.53 1.07 2.32 4.77 60.19 0.25 0.50 1.07 2.33 4.22 7 0.19 0.27 0.57 1.16 2.09 4.36 8 0.200.28 0.58 1.15 2.03 5.55 9 0.19 0.28 0.54 1.12 2.18 4.05 10  0.16 0.280.54 1.18 2.37 4.19 Replicate 1 0.20 0.20 0.44 0.89 1.94 4.05 2 0.200.23 0.44 0.93 2.05 4.10 3 0.21 0.24 0.42 0.93 2.17 3.91 4 0.20 0.250.45 0.88 2.03 4.20 5 0.22 0.21 0.47 0.94 1.98 3.92 6 0.21 0.25 0.400.96 2.06 3.95 7 0.20 0.19 0.41 0.88 2.12 4.14 8 0.20 0.24 0.43 1.022.05 3.88 9 0.19 0.28 0.46 1.00 2.07 4.10 10  0.19 0.21 0.50 0.96 2.173.80 Replicate 1 0.21 0.20 0.44 0.99 1.91 3.91 2 0.20 0.19 0.40 0.951.66 3.56 3 0.21 0.21 0.39 1.10 2.11 3.56 4 0.19 0.21 0.43 0.92 1.933.54 5 0.19 0.22 0.42 0.99 1.86 3.51 6 0.21 0.20 0.46 0.95 1.84 3.61 70.20 0.22 0.47 0.91 1.93 3.70 8 0.20 0.20 0.44 0.94 1.98 3.78 9 0.190.20 0.40 1.05 2.07 3.55 10  0.18 0.19 0.41 0.96 2.02 3.72 Replicate 10.18 0.26 0.45 0.89 1.60 3.85 2 0.21 0.25 0.48 0.82 1.52 4.17 3 0.220.26 0.50 0.89 1.51 3.75 4 0.21 0.28 0.48 0.86 1.52 3.74 5 0.21 0.280.44 0.92 2.20 3.66 6 0.19 0.24 0.43 0.86 1.74 3.87 7 0.18 0.25 0.470.92 1.62 3.86 8 0.21 0.24 0.43 0.88 1.52 3.63 9 0.19 0.28 0.43 0.871.51 3.67 10  0.21 0.23 0.46 0.91 1.56 3.98 Replicate 1 0.20 0.27 0.490.92 1.69 3.42 2 0.19 0.28 0.44 0.90 1.67 3.61 3 0.20 0.28 0.45 0.881.78 3.52 4 0.21 0.27 0.47 0.88 1.77 3.47 5 0.20 0.27 0.47 0.86 1.683.34 6 0.22 0.30 0.49 0.90 1.73 3.64 7 0.21 0.28 0.46 0.94 1.74 3.56 80.19 0.27 0.44 0.91 1.74 3.65 9 0.20 0.30 0.48 0.88 1.66 3.31 10  0.170.31 0.46 0.87 1.67 3.40 Average 0.20 0.25 0.46 0.96 1.91 3.89 SD 0.010.03 0.04 0.10 0.25 0.41 RSD (%) 6.3 13.4 9.3 9.9 13.1 10.6 Accuracy (%)104.0 99.3 92.6 96.2 95.5 97.2

Analysis of data for γ-tocopherol spiked serum samples (shown in Table4) shows that a concentration range of about 0.09 mg/L to about 1.95mg/L yields relative standard deviations of 11.1% to 22.4% andaccuracies of about 64.7% to about 101.3%. Acceptable reproducibilityconcentrations (RSD≤20%) are observed at 0.12 mg/L and higher. The RSDand accuracy at low concentration levels is shown in FIG. 8.

TABLE 4 LLOQ Data for γ-Tocopherol Spiked Serum Samples γ-TocopherolStandard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Actual(mg/L) 0.09 0.12 0.24 0.49 0.98 1.95 Replicate 1 0.05 0.13 0.22 0.480.99 1.84 2 0.08 0.13 0.24 0.47 1.05 1.85 3 0.06 0.11 0.21 0.50 0.942.01 4 0.05 0.11 0.22 0.47 1.02 1.92 5 0.05 0.11 0.25 0.47 1.00 1.92 60.08 0.11 0.23 0.50 1.01 1.93 7 0.05 0.12 0.26 0.51 1.03 1.72 8 0.070.13 0.26 0.46 0.91 2.09 9 0.05 0.13 0.27 0.55 0.93 1.94 10  0.04 0.120.26 0.42 1.05 2.29 Replicate 1 0.06 0.09 0.22 0.39 0.94 1.82 2 0.050.12 0.18 0.41 0.90 1.58 3 0.07 0.13 0.18 0.38 1.03 1.65 4 0.05 0.140.20 0.41 0.94 2.06 5 0.04 0.07 0.22 0.43 0.95 1.74 6 0.06 0.07 0.220.44 1.01 1.82 7 0.07 0.12 0.21 0.40 0.84 1.92 8 0.06 0.13 0.19 0.460.89 1.67 9 0.05 0.11 0.18 0.43 0.89 1.70 10  0.05 0.11 0.21 0.43 0.921.66 Replicate 1 0.06 0.10 0.20 0.43 1.03 1.89 2 0.07 0.07 0.23 0.571.04 1.71 3 0.06 0.10 0.22 0.63 1.19 1.69 4 0.06 0.06 0.23 0.54 1.281.78 5 0.06 0.06 0.22 0.53 1.10 1.80 6 0.04 0.10 0.21 0.53 1.22 1.95 70.05 0.08 0.28 0.55 1.28 2.28 8 0.04 0.09 0.21 0.54 1.17 1.77 9 0.070.07 0.29 0.61 1.13 2.67 10  0.04 0.07 0.20 0.60 1.18 2.50 Replicate 10.07 0.11 0.26 0.58 0.92 2.38 2 0.06 0.12 0.23 0.50 0.80 1.97 3 0.060.12 0.29 0.59 0.87 1.80 4 0.04 0.12 0.26 0.59 1.02 1.82 5 0.05 0.120.30 0.65 1.08 1.83 6 0.07 0.12 0.29 0.60 0.93 1.78 7 0.04 0.12 0.280.59 1.03 1.83 8 0.03 0.11 0.26 0.58 0.90 1.79 9 0.06 0.12 0.21 0.551.08 1.86 10  0.05 0.12 0.30 0.57 0.95 1.86 Replicate 1 0.06 0.14 0.230.40 0.92 1.77 2 0.06 0.13 0.22 0.53 0.93 1.87 3 0.06 0.12 0.22 0.410.89 1.84 4 0.07 0.11 0.22 0.45 0.98 1.72 5 0.05 0.12 0.24 0.42 0.881.78 6 0.07 0.08 0.22 0.49 0.97 1.93 7 0.08 0.09 0.24 0.41 0.88 1.87 80.09 0.13 0.19 0.43 0.90 1.94 9 0.08 0.11 0.18 0.48 0.93 1.78 10  0.070.09 0.22 0.47 0.94 1.39 Average 0.06 0.11 0.23 0.50 0.99 1.88 SD 0.010.02 0.03 0.07 0.11 0.22 RSD (%) 22.4 20.0 14.2 14.7 11.1 11.9 Accuracy(%) 64.7 89.8 96.5 101.3 101.3 96.4

The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as three standarddeviations from lowest measurable concentration. To determine the LODfor vitamin A, α-tocopherol, and γ-tocopherol, ten spiked stripped serumsamples at the LLOQ level were assayed, and the results analyzed. TheLOD for vitamin A was determined to be 0.38 mcg/dL, while the LODs forα-tocopherol and γ-tocopherol were both determined to be 0.03 mg/L.

Example 5: Linearity of Detection for Vitamin A, α-Tocopherol, andγ-Tocopherol

Three separate assays, each including nine standards at variousconcentrations of each of vitamin A, α-tocopherol, and γ-tocopherol(ranging from about 0.74 mcg/dL to about 172.00 mcg/dL for vitamin A,about 0.24 mg/L to about 60.00 mg/L for α-tocopherol, and about 0.12mg/L to about 31.20 mg/L for γ-tocopherol). Linear graphs wereconstructed using linear regression without weighting. All analytesexhibited regression coefficients (R² values) of greater than 0.9995.Plots of peak area ratios versus respective target values demonstratingthe linearity of response are shown in FIGS. 9 and 10 for vitamin A,α-tocopherol, and γ-tocopherol, respectively.

Example 5: Intra- and Inter-Assay Variation and Accuracy for Vitamin A,α-Tocopherol, and γ-Tocopherol

Intra-assay variation is defined as the reproducibility of analysis of asample within an assay. The RSD for the replicates of the sample atthree levels were used to determine if the reproducibility is acceptable(i.e., ≤15% RSD). Twenty replicates from each of three levels wereanalyzed for each of the analytes (about 7.00 mcg/dL, about 30.00mcg/dL, and about 100.00 mcg/dL for vitamin A; about 2.50 mg/L, about10.00 mg/L, and about 22 mg/L for α-tocopherol; and about 1.50 mg/L,about 6.00 mg/L, and about 12.00 mg/L for γ-tocopherol). Data from theseanalyses are presented in Tables 5-7. The intra-assay variation RSDs ofthe three analytes were determined to be about 4.3%-6.7% for vitamin A,about 3.1%-6.1% for α-tocopherol, and about 4.1%-6.3% for γ-tocopherol.

Intra-assay accuracy is defined as the accuracy of measurement within anassay. The acceptable range of intra-assay accuracy is between 85%-115%.Twenty replicates of the three sample levels described above for eachanalyte were analyzed to demonstrate that the intra-assay accuracies ofall three analytes are within the range of about 96.1% to about 102.9%.Results of data analysis are shown in Tables 5, 6, and 7.

TABLE 5 Intra-assay Variation Data and Results for Vitamin A Vitamin ALow Medium High 7.00 30.00 100.00 Replicate mcg/dL mcg/dL mcg/dL 1 7.3931.25 104.45 2 7.24 31.20 100.24 3 6.68 27.28 103.83 4 6.67 30.12 102.955 6.78 29.41 93.85 6 7.03 31.45 105.87 7 7.24 28.00 105.73 8 7.34 28.05105.55 9 7.07 26.33 102.81 10 6.68 30.66 109.32 11 6.45 30.32 106.44 127.54 32.23 105.22 13 6.56 32.16 101.47 14 6.84 28.61 105.68 15 7.7226.60 108.95 16 7.81 26.74 96.34 17 6.56 26.86 99.76 18 7.12 28.24 94.0019 6.62 28.00 105.51 20 6.26 30.48 99.58 Mean 6.98 29.20 102.88 SD 0.441.96 4.42 RSD (%) 6.3 6.7 4.3 Accuracy (%) 99.7 97.3 102.9

TABLE 6 Intra-assay Variation Data and Results for α-Tocopherolα-Tocopherol Low Medium High 2.50 10.00 22.00 Replicate mg/L mg/L mg/L 12.56 9.70 22.49 2 2.72 9.64 22.64 3 2.70 9.75 21.65 4 2.62 10.01 23.87 52.73 9.61 23.90 6 2.53 9.43 23.17 7 2.69 9.52 23.98 8 2.34 10.21 22.81 92.48 9.60 23.05 10 2.56 10.07 22.05 11 2.58 9.80 22.31 12 2.25 9.7922.67 13 2.24 11.11 21.78 14 2.69 9.32 23.08 15 2.67 9.65 22.85 16 2.699.64 22.15 17 2.43 9.59 22.10 18 2.71 9.64 22.35 19 2.38 9.50 21.84 202.59 9.82 21.99 Mean 2.56 9.77 22.64 SD 0.16 0.38 0.71 RSD (%) 6.1 3.93.1 Accuracy (%) 102.3 97.7 102.9

TABLE 7 Intra-assay Variation Data and Results for γ-Tocopherolγ-Tocopherol Low Medium High 1.50 6.00 12.00 Replicate mg/L mg/L mg/L 11.49 5.62 11.52 2 1.68 6.04 11.96 3 1.54 5.44 11.42 4 1.59 5.50 12.45 51.45 5.68 12.74 6 1.57 5.69 12.25 7 1.49 5.77 12.51 8 1.48 5.77 11.98 91.46 5.92 12.23 10 1.48 5.66 12.39 11 1.41 5.97 12.25 12 1.35 5.46 10.0813 1.32 6.40 12.63 14 1.39 5.89 12.05 15 1.43 6.11 11.87 16 1.41 5.7412.52 17 1.40 5.65 12.97 18 1.57 5.52 12.45 19 1.52 5.86 11.99 20 1.335.68 12.52 Mean 1.47 5.77 12.14 SD 0.09 0.24 0.62 RSD (%) 6.3 4.1 5.1Accuracy (%) 97.9 96.1 101.2

The inter-assay variation is defined as the reproducibility (RSD) of asample between assays. The acceptable precision requirement forinter-assay study is ≤15% RSD. Ten replicates of the three sample levelsdescribed above for each analyte were analyzed on five different days.Data from these analyses are shown in Tables 8, 9, and 10. Theinter-assay variation (RSD) was determined to be about 5.2%-8.1% forvitamin A, about 3.1%-6.1% for α-tocopherol, and about 4.1%-6.3% forγ-tocopherol.

Inter-assay accuracy is defined as the accuracy of measurement betweenassays. The acceptable range of inter-assay accuracy is between85%-115%. The same ten replicates of the three sample levels describedabove for each analyte were used to demonstrate that the intra-assayaccuracies of all three analytes are within the range of about 97.4% toabout 104.9%. Results of data analysis are shown in Tables 8, 9, and 10.

TABLE 8 Inter-assay Variation Data and Results for Vitamin A Vitamin ALow Medium High 7.00 30.00 100.00 Replicate mcg/dL mcg/dL mcg/dL Day 1,Replicate 1 7.73 30.94 106.17 2 7.67 28.92 107.92 3 7.45 29.61 106.47 48.17 32.30 105.69 5 6.95 31.33 98.99 6 8.09 30.35 106.74 7 7.25 29.59106.13 8 8.27 30.36 105.79 9 7.69 29.63 102.00 10  8.82 29.48 100.04 Day2, Replicate 1 7.77 27.37 109.49 2 7.12 32.10 95.48 3 7.16 26.80 102.844 8.54 30.34 101.40 5 7.25 30.17 94.33 6 8.18 28.57 92.58 7 6.57 28.2089.54 8 7.10 32.59 103.00 9 7.57 28.13 86.47 10  6.97 26.85 92.98 Day 3,Replicate 1 7.29 31.07 101.74 2 7.72 33.95 108.52 3 7.76 29.76 107.71 47.56 31.17 103.01 5 7.95 32.28 104.53 6 8.17 33.30 106.64 7 8.32 27.56101.57 8 7.64 27.95 102.68 9 6.73 28.02 100.84 10  7.49 29.70 104.00 Day4, Replicate 1 7.09 30.17 97.42 2 7.27 26.03 99.47 3 6.42 28.34 97.80 47.36 26.46 100.08 5 6.68 25.05 111.06 6 6.97 26.24 107.80 7 7.33 26.95102.74 8 6.35 26.42 98.35 9 6.07 26.29 104.88 10  6.59 26.49 98.88 Day5, Replicate 1 7.39 31.25 104.45 2 7.24 31.20 100.24 3 6.68 27.28 103.834 6.67 30.12 102.95 5 6.78 29.41 93.85 6 7.03 31.45 105.87 7 7.24 28.00105.73 8 7.34 28.06 105.55 9 7.07 26.33 102.81 10  6.68 30.66 109.32Mean 7.34 29.21 102.17 SD 0.60 2.15 5.27 RSD (%) 8.1 7.4 5.2 Accuracy(%) 104.9 97.4 102.2

TABLE 9 Inter-assay Variation Data and Results for α-Tocopherolα-Tocopherol Low Medium High 2.50 10.00 22.00 Replicate mg/L mg/L mg/LDay 1, Replicate 1 2.43 9.72 22.44 2 2.51 9.39 22.08 3 2.45 9.74 22.38 42.56 9.24 22.75 5 2.80 9.80 22.76 6 2.91 9.58 22.15 7 2.53 9.50 22.61 82.37 9.53 22.42 9 2.45 11.03 22.16 10  2.52 10.77 22.46 Day 2, Replicate1 2.41 9.60 22.78 2 2.45 10.49 20.77 3 2.41 9.58 22.74 4 2.37 9.20 22.105 2.83 9.33 22.65 6 2.80 9.82 21.83 7 2.51 9.84 22.59 8 2.37 10.52 21.869 2.52 10.61 19.68 10  2.53 10.59 20.59 Day 3, Replicate 1 2.39 10.6322.75 2 2.48 9.34 22.56 3 2.52 9.21 21.74 4 2.39 9.75 22.18 5 2.97 9.2622.85 6 2.71 10.04 22.64 7 2.34 11.47 22.39 8 2.46 11.23 21.02 9 2.5511.07 22.35 10  2.32 11.26 21.35 Day 4, Replicate 1 2.13 9.15 22.31 22.17 8.93 23.19 3 2.20 9.05 23.48 4 2.15 8.91 23.42 5 2.09 9.73 23.47 62.13 9.58 23.32 7 2.03 9.83 22.09 8 2.06 9.68 23.33 9 2.32 9.75 22.3610  2.06 9.70 22.49 Day 5, Replicate 1 2.72 9.64 22.64 2 2.70 9.75 21.653 2.62 10.01 23.87 4 2.73 9.61 23.90 5 2.53 9.43 23.17 6 2.69 9.52 23.997 2.34 10.21 22.81 8 2.48 9.60 23.05 9 2.56 10.07 22.05 10  2.58 9.8022.01 Mean 2.46 9.86 22.44 SD 0.22 0.63 0.83 RSD (%) 9.1 6.4 3.7Accuracy (%) 98.5 98.6 102.0

TABLE 10 Inter-assay Variation Data and Results for γ-Tocopherolγ-Tocopherol Low Medium High 1.50 6.00 12.00 Replicate mg/L mg/L mg/LDay 1, Replicate 1 1.72 5.58 11.05 2 1.41 5.43 11.80 3 1.56 5.51 11.22 41.52 5.44 11.64 5 1.53 6.24 11.48 6 1.64 6.98 11.65 7 1.62 6.57 11.74 81.66 6.57 11.54 9 1.49 6.91 11.68 10  1.46 5.81 11.50 Day 2, Replicate 11.51 5.55 12.71 2 1.41 5.65 11.51 3 1.48 5.56 10.99 4 1.49 5.49 11.39 51.45 5.49 11.57 6 1.59 6.49 12.55 7 1.58 6.25 12.40 8 1.53 6.59 12.68 91.53 6.12 12.38 10  1.54 6.62 11.82 Day 3, Replicate 1 1.49 6.40 11.80 21.46 5.05 10.33 3 1.41 6.25 10.35 4 1.42 5.80 11.23 5 1.70 6.08 12.02 61.59 6.92 12.65 7 1.40 6.82 11.26 8 1.39 5.37 11.12 9 1.48 5.59 11.4010  1.38 5.72 10.87 Day 4, Replicate 1 1.36 5.38 12.42 2 1.19 5.51 13.153 1.42 6.04 12.97 4 1.40 5.56 12.25 5 1.23 5.63 11.65 6 1.21 5.71 12.447 1.24 5.67 11.60 8 1.25 5.97 11.91 9 1.30 5.56 11.77 10  1.25 6.0511.98 Day 5, Replicate 1 1.49 5.62 11.52 2 1.68 6.04 11.96 3 1.54 5.4411.42 4 1.59 5.50 12.45 5 1.45 5.68 12.74 6 1.56 5.69 12.25 7 1.49 5.7712.41 8 1.48 5.77 11.98 9 1.47 5.92 12.23 10  1.41 5.97 12.39 Mean 1.475.91 11.84 SD 0.13 0.47 0.62 RSD (%) 8.6 8.0 5.3 Accuracy (%) 97.9 98.498.6

Example 6: Matrix Specificity for Vitamin A, α-Tocopherol, andγ-Tocopherol

The effects of different matrices on the quantitation of vitamin A,α-tocopherol, and γ-tocopherol were studied with duplicate samples. Oneserum sample with high concentration vitamin A, α-tocopherol, andγ-tocopherol was diluted in duplicate from 2.5× to 50× with Biocelldouble stripped serum, deionized water, and normal saline. The studyindicated that Biocell serum and normal saline could be used fordilution up to 50×. Inaccurate values for vitamin A were determined forhigh dilutions with deionized water.

Example 7: Recovery Studies for Vitamin A, α-Tocopherol, andγ-Tocopherol

A recovery study of spiked vitamins A, α-tocopherol, and γ-tocopherol indouble stripped Biocell serum was performed in duplicates at eightdifferent concentration levels for each analyte. All three analytes werequantitated simultaneously for each sample. Recovery was calculatedusing obtained values versus target values. The study yielded totalrecovery as 97.8%, 101.2%, and 93.4% for vitamin A, α-tocopherol, andγ-tocopherol, respectively.

Example 8: Interference Studies for Vitamin A, α-Tocopherol, andγ-Tocopherol

The interference effects of other vitamins and related compounds at highconcentrations, as well as the effects of drugs at concentrations of 10mcg/mL, were studied. None of the tested vitamins and related compoundsor drugs demonstrated interference effects at the concentrations tested.A listing of the compounds tested and their concentrations are shown inTables 11 and 12.

TABLE 11 Interference of Vitamins and Related Compounds AnalytesConcentration (mcg/mL) Interference Vitamin K1 10 No Vitamin K2 10 NoVitamin B6 10 No Vitamin B12 10 No Folic Acid 10 No α and β-Carotenes 10No Vitamin B2 10 No Vitamin B1 10 No Pyrithiamin 10 No Folic Acid 10 NoVitamin D2 800 nmol No Vitamin D3 800 nmol No

TABLE 11 Interference of Vitamins and Related Compounds AnalytesConcentration (mcg/mL) Interference Imipramine 10 No Desipramine 10 NoAmitriptyline 10 No Nortriptyline 10 No Doxepin 10 No N-Desmethyldoxepin10 No Fluoxetine 10 No N-desmethylfluoxetine 10 No Maprotiline 10 NoMycophenolic Acid 10 No Mycophenolic Acid Glucoronide 10 No Propanolol10 No Clomipramine 10 No N-Desmethylclomipramine 10 No Felbamate 10 NoRapamycine 10 No Cyclosporine 10 No Gabapentin 10 No Zonisamide 10 NoLidocaine 10 No

Example 8: Comparison of MS/MS Method and HPLC Method for β-Tocopheroland γ-Tocopherol

Results from analysis of patient samples with the above described MS/MSmethod were correlated to results from an HPLC method that is known tobe unable to distinguish between β-tocopherol and γ-tocopherol.

The R² value from these correlation studies was 0.9742. Thisdemonstrates that the MS/MS method described in the Examples above isalso unable to distinguish between β-tocopherol and γ-tocopherol, withthe resulting quantitation reflecting the combined amounts ofβ-tocopherol and γ-tocopherol in the samples.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining the amount ofvitamin A in a sample by tandem mass spectrometry, said methodcomprising: a. subjecting vitamin A from a sample to ionization underconditions suitable to produce one or more ions detectable by massspectrometry; and b. determining the amount of one or more of said ionsby tandem mass spectrometry, wherein tandem mass spectrometry comprisesfragmenting a vitamin A precursor ion having a mass to charge ratio ofabout 269.30±0.80 into one or more fragment ions comprising a fragmention having a mass to charge ratio of about 105.00±0.80; wherein theamount of the one or more fragment ions determined in step (b) isrelated to the amount of vitamin A in the sample.
 2. The method of claim1 wherein the sample is subjected to liquid chromatography (LC) prior toionization.
 3. The method of claim 2, wherein said LC is reverse phaseLC.
 4. The method of claim 2 wherein said LC and said ionization areconducted with on-line processing.
 5. The method of claim 1 wherein thesample is subjected to high performance liquid chromatography (HPLC)prior to ionization.
 6. The method of claim 1, wherein said ionizationcomprises ionization with an atmospheric chemical ionization (APCI)source.
 7. The method of claim 1, wherein the sample comprises abiological sample.
 8. The method of claim 7, wherein the biologicalsample is from a human patient suspected of having a deficiency or anexcess of vitamin A.
 9. The method of claim 1, wherein the samplecomprises serum or plasma.
 10. The method of claim 1, wherein tandemmass spectrometry is conducted by multiple reaction monitoring,precursor ion scanning, or product ion scanning.
 11. The method of claim1 further comprising determining the amount of one or more additionalanalytes simultaneously with the determination of the amount of vitaminA.
 12. The method of claim 11, wherein the one or more additionalanalytes comprises one or both of α-tocopherol and combined β-tocopheroland γ-tocopherol.
 13. The method of claim 12 wherein the one or moreadditional analytes comprises α-tocopherol, and determining the amountof α-tocopherol comprises fragmenting an α-tocopherol precursor ionhaving a mass to charge ratio of about 430.47±0.80 into one or morefragment ions comprising a fragment ion having a mass to charge ratio ofabout 165.03±0.80.
 14. The method of claim 12 wherein the one or moreadditional analytes comprises combined β-tocopherol and γ-tocopherol,and determining the amount of combined β-tocopherol and γ-tocopherolcomprises fragmenting β-tocopherol and γ-tocopherol precursor ionshaving a mass to charge ratio of about 416.35±0.80 into one or morefragment ions comprising a fragment ion having a mass to charge ratio ofabout 151.00±0.80.
 15. The method of claim 12 wherein the one or moreadditional analytes comprises α-tocopherol and combined β-tocopherol andγ-tocopherol.